Anti-histone H1 monoclonal antibody and hybridoma for the production thereof

ABSTRACT

The present invention discloses anti-histone H1 monoclonal antibodies, hybridomas for the production thereof, and polypeptides, which are useful for suppressing, predicting, or diagnosing transplant rejection in organ transplantation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/661,763, filed Apr. 3, 2007, which is a national stage application ofInternational application No. PCT/JP2005/016268, filed Sep. 5, 2005.

This application claims the benefit of priority from the prior JapanesePatent Application No. 2004-257528 filed on Sep. 3, 2004, the entiredisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an anti-histone H1 monoclonal antibody,a hybridoma for the production thereof, and a polypeptide which theanti-histone H1 antibody specifically recognizes. More specifically, thepresent invention relates to a monoclonal antibody, a hybridomaproducing thereof, and polypeptide, which are useful for suppressing,predicting, or diagnosing transplant rejection in organ transplantation.

2. Background Technology

In organ transplantation medicine, in order to suppress transplantrejection after organ transplantation, various immunosuppressive agentshave so far been used. Examples of such immunosuppressive agents includetacrolimus (FK506) and cyclosporin A (Jpn J Pharmacol, 71, 89-100,1996). However, conventional immunosuppressive agents have problems suchas strong side effects including promotion of the growth of cancer cellsand suppression of bone marrow functions, infections, and need forlong-lasting administration (Transplantation, 58, 170-178, 1994).

Further, it is generally difficult to assess the time to withdrawimmunosuppressive agents. For example, transplanted tissue occasionallysurvives without continuing the administration of an immunosuppressiveagent. In such cases, if the administration of an immunosuppressiveagent is carelessly continued, damages simply due to its toxicity may bedone to a patient. On the other hand, it is also possible that survivingtissue becomes rejected by discontinuing the administration of animmunosuppressive agent. In this case, the rejection often cannot beevaded by restarting the administration of an immunosuppressive agent.

On the other hand, various studies on organ transplantation have beencarried out. For example, in the system of orthotopic livertransplantation (OLT), it has been reported that when the liver of adonor DA rat (MHC haplotype, RT1a), which has a high graft survivalrate, was transplanted into a recipient PVG rat (RT1c), the graftsurvived without administering an immunosuppressive agent(Transplantation, 35, 304-311, 1983).

Further, it has been reported that transplant graft rejection issuppressed in a transplantation model system related to a combinationwhich generates transplant rejection by administering once prior tooperation the serum of a recipient PVG rat into which the liver of DArat was transplanted (post-OLT serum) to the system (J. Surg. Res., 80,56-61, 1998).

Further, it has been disclosed that transplant rejection is suppressedand a recipient survives in a heart transplantation system of a DA rat(RT1a) and a LEWIS rat (RT1l) (in vivo), which always generatestransplant rejection by administering an anti-histone H1 polyclonalantibody to the system to after operation (Transplantation, 77,1595-1603, 2004).

Further, some of the present inventors have disclosed that mixedlymphocyte reaction (MLR) is suppressed by using serum derived from aPVG rat in the early stage after transplantation and that ananti-histone H1 antibody has an MLR suppressive activity (JapanesePatent Laid-open Publication No. 2004-149507).

However, the development of a novel immunosuppressive agent which cansuppress transplant rejection in organ transplantation and is excellentin safeness and its immunosuppressive activity still has been desired.Further, since it is necessary to monitor prognosis of patients or toprevent unnecessary administration of immunosuppressive agents in organtransplantation, the development of a novel drug with excellent accuracyfor predicting or diagnosing the incidence of transplant rejection hasalso been desired.

SUMMARY OF THE INVENTION

The present inventors have now found an anti-histone H1 monoclonalantibody which has a marked immunosuppressive activity and is useful insuppressing, predicting, and diagnosing transplant rejection, and ahybridoma which produces this antibody. Further, the present inventorshave found a specific amino acid sequence which is specificallyrecognized by the abovementioned anti-histone H1 monoclonal antibody.The present invention is based on these findings.

Accordingly, an objective of the present invention is to provide ananti-histone H1 monoclonal antibody which has a marked immunosuppressiveactivity and is useful in suppressing, predicting, or diagnosingtransplant rejection and a hybridoma which produces this antibody.

Further, an objective of the present invention is to provide apolypeptide comprising a specific amino acid sequence which isspecifically recognized by the abovementioned anti-histone H1 monoclonalantibody.

The monoclonal antibody according to the present invention recognizeshistone Hi or a histone H1-like antigen present in the cell membrane ofa spleen cell.

Further, the hybridoma according to the present invention produces theabovementioned monoclonal antibody.

Further, the polypeptide according to the present invention comprises aspecific amino acid sequence which is recognized by the monoclonalantibody according to the present invention.

The monoclonal antibody according to the present invention has a markedimmunosuppressive activity and safeness and can be advantageously usedas an excellent immunosuppressive agent. Further, the monoclonalantibody according to the present invention has an excellent specificityto an autoantigenic protein in a mammal which makes an index oftransplant rejection, so that it can be advantageously used forpredicting or diagnosing transplant rejection in organ transplantationof a mammal.

Further, the polypeptide according to the present invention can beadvantageously used as an immunosuppressive agent since it can inducethe production of an anti-histone H1 antibody in the living body whenused as an antigen. Furthermore, the polypeptide according to thepresent invention can be used for measuring the amount of anti-histoneH1 antibody produced in a mammal, so that it can be advantageously usedfor predicting or diagnosing transplant rejection in organtransplantation.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the result of the evaluation for the MLR-suppressingactivity using culture supernatants containing an anti-histone H1monoclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION Deposition

The hybridomas according to the present invention, i.e., hybridoma 1F5,hybridoma 3F2, hybridoma 15F11, hybridoma 17C2, and hybridoma 16G9, haveoriginally been deposited with the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) on Aug.19, 2004, under Accession Numbers FERM ABP-10409, FERM ABP-10410, FERMABP-10411, FERM ABP-10412, and FERM ABP-10413, respectively.

Monoclonal Antibodies and Hybridomas

A monoclonal antibody according to the present invention recognizeshistone H1 or a histone H1-like antigen present in a spleen cell.Further, the monoclonal antibody according to the present inventionrecognizes an epitope in an amino acid sequence preferably representedby SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:8. Further, according to another preferred embodiment of the presentinvention, the anti-histone H1 monoclonal antibody recognizes apolypeptide comprising an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Saidpolypeptide preferably consists of about 12-150 amino acid residues.Further, according to another preferred embodiment of the presentinvention, the anti-histone H1 monoclonal antibody recognizes apolypeptide consisting of an amino acid sequence represented by SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.Further, according to another preferred embodiment of the presentinvention, the anti-histone H1 monoclonal antibody is produced by one ormore hybridomas selected from the group consisting of hybridoma 1F5,hybridoma 3F2, hybridoma 15F11, hybridoma 17C2, and hybridoma 16G9.

The term “histone H1” refers to a basic protein which is present in aeukaryotic cell and binds nucleosome linker DNA to form a nucleosome.Examples of such histone H1 include one which is derived from a humanand comprises an amino acid sequence represented by SEQ ID NO: 1, onewhich is derived from cattle and comprises an amino acid sequencerepresented by SEQ ID NO: 2, and one which is derived from a mouse andcomprises an amino acid sequence represented by SEQ ID NO: 3.

The term “histone H1-like antigen” refers to an antigen which isrecognized by a monoclonal antibody produced by hybridoma 1F5, hybridoma3F2, hybridoma 15F11, hybridoma 17C2, or hybridoma 16G9 in the cellmembrane of a spleen cell. This histone H1-like antigen preferablyconstitutes a part of a protein having a molecular weight of 31 kD onSDS-PAGE. An example of such histone H1-like antigen is a protein havingat least a part of the amino acid sequence of histone H1 derived from amammal.

Further, the monoclonal antibody according to the present invention maybe a chimeric antibody, a humanized antibody, or a complete humanizedantibody, if desired. More specific examples include a chimeric antibodyin which an antigen binding domain Fv of a mouse monoclonal antibody isalternatively introduced into a human antibody (Morrison, S. L., Oi, V.T., “Immunoglobulin Genes” Academic Press (London), 260-274 (1989)) anda humanized antibody in which a complementary determining region (CDR)that is a sequence on an Fv domain directly involved in the antigenbinding of a mouse monoclonal antibody is embedded into a human antibodyframe using CDR graft technology (Roguska, M. L. et. al., Humanizationof murine monoclonal antibodies through variable domain resurfacing,Proc. Natl. Acad. Sci. USA, 91, 969-973 (1994)). Further, examples ofthe complete humanized antibody include one produced by the TransChromoMouse to which a human antibody gene is transplanted (Tomizuka, K. et.al., Functional expression and germline transmission of a humanchromosome fragment in chimaeric mice, Nature Genet., 16, 133-143(1997)), or one produced by human antibody phage libraries (Winter, G.et. al., Making antibodies by phage display technology, Ann. Rev.Immunol., 12, 433-455 (1994); Griffiths, A. D. et. al., Isolation ofhigh affinity human antibodies directly from large syntheticrepertoires, EMBO. J., 13, 3245-3260 (1994)).

Further, according to another embodiment of the present invention, thereis provided hybridoma 1F5, hybridoma 3F2, hybridoma 15F11, hybridoma17C2, and hybridoma 16G9.

The monoclonal antibody and hybridoma according to the present inventioncan be manufactured, for example, as follows. Namely, first, thehybridoma according to the present invention can be obtained by usinghistone H1 or a histone H1-like antigen or a polypeptide containing anepitope thereof as a sensitizing antigen, fusing a mammalian plasma cellof a mammal immunized with this sensitizing antigen (immunocyte), with amammalian myeloma cell, cloning the obtained hybridomas and selectingdesired hybridomas from them. The monoclonal antibody according to thepresent invention can be obtained by culturing the hybridoma accordingto the present invention so as to recover the antibody produced by it.

The polypeptide containing histone H1 or a histone H1-like antigen or anepitope thereof used as sensitizing antigen can be derived, for example,from human leukemic bone marrow cells, human cervix uteri cancer Helacells, bovine thymus gland, bovine liver, bird erythrocytes, and thelike. This sensitizing antigen is suspended, for example, in PBS orphysiological saline, and the suspension is used for immunization of amammal with an adjuvant such as FCA (Freund's complete adjuvant) and KLH(keyhole limpet hemocyanin), if desired.

As a method of immunizing a mammal, a general administration method inthis field of technology can be used. Specific examples of theadministration method include intraperitoneal injection, intrasplenicinjection intramuscular injection, subcutaneous injection,intracutaneous injection, oral administration, mucosal administration,and intradermal administration, preferably intraperitoneal injection andintrasplenic injection. The interval between administrations of thesensitizing antigen is appropriately determined depending on the dose ofthe sensitizing antigen, the kind of the mammal, and the like; forexample, it can be administered several times a month.

The mammals to be immunized are not particularly limited; however, theyare preferably selected taking into consideration compatibility with themyeloma cell used in cell fusion; examples include mice, rats, andhamsters, preferably mice.

Further, spleen cells are preferably used as immunizing cells.

Examples of the myeloma cell used in the present invention include P3(P3X63Ag8.653) (J. Immunol., 123, 1548, 1978), p3-U1 (Current Topics inMicrobiology and Immunology, 81, 1-7, 1978), NS-1 (Eur. J. Immunol., 6,511-519, 1976), MPC-11 (Cell, 8, 405-415, 1976), Sp2/0-Ag14 (Nature,276, 269-270, 1978), FO (J. Immunol. Meth., 35, 1-21, 1980), S194 (J.Exp. Med., 148, 313-323, 1978), and R210 (Nature, 277, 131-133, 1979),preferably P3 or p3-U1, and more preferably P3.

The cell fusion between an immunizing cell and a myeloma cell can becarried out, for example, according to the method of Milstein et al.(Methods Enzymol., 73, 3-46, 1981). More specifically, the cell fusioncan be carried out, for example, by mixing the immunizing cell and themyeloma cell in a medium in the presence of a fusion promoting agent. Incell fusion, hybridomas can be produced by appropriately repeating theaddition of medium and centrifugation.

The medium for cell fusion can be, for example, a medium generally usedfor cell fusion, such as RPMI-1640 medium and MEM medium. Further, serumsupplements such as fetal bovine serum (FBS) can be appropriately usedtogether.

Further, the cell fusion is carried out preferably at 25-37° C., morepreferably at 30-37° C.

The mixing ratio of the myeloma cell and the immunizing cell ispreferably about 1:1-1:10.

Examples of the fusion promoting agent include polyethylene glycol (PEG)and Sendai virus (HVJ), preferably PEG. The molecular weight of PEG canbe appropriately selected; for example, the average molecular weight ofPEG can be about 1,000 to 6,000. Further, the concentration of PEG in amedium is preferably about 30-60% (W/V).

Furthermore, if necessary, an auxiliary agent such as dimethyl sulfoxideis appropriately added to a medium.

Selection of a hybridoma according to the present invention can becarried out by culturing hybridomas obtained by cell fusion in a normalselective medium such as an HAT medium and performing screening by ageneral limiting dilution method, for example, using antibody titer tohistone H1 as an index. The time for cultivation in the HAT medium isenough to kill cells (non-fused cells) other than a target hybridoma andcan generally be several days to several weeks. A hybridoma according tothe present invention thus obtained can be subcultured in a normalmedium and stored in liquid nitrogen for a long period of time.

Further, examples of the method of recovering a monoclonal antibodyaccording to the present invention include a method in which hybridomasare cultured by a normal method to obtain a monoclonal antibody from theresulting culture supernatant and a method in which hybridomas areadministered to a compatible mammal for proliferation to obtain amonoclonal antibody from abdominal fluid of this mammal. The formermethod is preferred to obtain an antibody with a high purity, while thelatter is preferred to produce an antibody in large quantities.

Furthermore, the monoclonal antibody according to the present inventioncan be purified to high purity using a salting-out method, a gelfiltration method, affinity chromatography, and the like.

The monoclonal antibody according to the present invention has a markedimmunosuppressive activity as mentioned above. The monoclonal antibodyaccording to the present invention can be used as it is as animmunosuppressive agent; however, it can also be used as apharmaceutical composition, particularly as a composition forsuppressing immunity, along with pharmaceutically acceptable carriersand the like. Therefore, according to an embodiment of the presentinvention, there is provided a composition for suppressing immunitycomprising the monoclonal antibody according to the present invention asactive ingredient. According to another embodiment of the presentinvention, there is also provided use of the monoclonal antibodyaccording to the present invention in manufacturing the composition forsuppressing immunity.

The composition for suppressing immunity according to the presentinvention is useful for the treatment and prevention of transplantrejection in the transplantation of organs such as the heart, kidney,liver, bone marrow, and skin, further for the treatment and preventionof autoimmune diseases. The composition for suppressing immunityaccording to the present invention can be prepared, for example, bydissolving the monoclonal antibody according to the present invention inphysiological saline for injection, distilled water for injection, abuffer solution for injection, and the like. Further, the compositionfor suppressing immunity according to the present invention can containappropriate solvents, solubilizing agents, preservatives, stabilizers,emulsifiers, suspending agents, analgesic agents, isotonizing agents,buffering agents, fillers, thickening agents, coloring agents, and knowncarriers (e.g., various liposomes, polyamino acid carriers, syntheticpolymers, natural polymers).

The composition for suppressing immunity according to the presentinvention can be administered either systemically or locally. Specificexamples of the method of administration include dripping, intravenousinjection, intramuscular injection, subcutaneous injection, intradermalinjection, oral administration, mucosal administration, and transdermaladministration. Therefore, according to another embodiment of thepresent invention, there is provided a method for treating a mammalwhich requires immunosuppression, comprising the administering atherapeutically effective amount of a monoclonal antibody according tothe present invention to a mammal. The dose of the monoclonal antibodyaccording to the present invention depends on the condition and age ofthe mammal and the like; however, generally, it can be administered adose of 0.05-40 mg/kg bodyweight/day, preferably 0.1-1.0 mg/kgbodyweight/day in a single or several divided doses. Further, theadministration can be a single or, for example, repeated over the periodof 4 weeks.

Further, the monoclonal antibody according to the present invention canbe used in predicting or diagnosing transplant rejection in a mammalsince it specifically reacts with an auto-antigenic protein which is anindex for transplant rejection upon organ transplantation. Theauto-antigenic protein herein means a protein which is present in themammal and preferably recognized by a monoclonal antibody produced byhybridoma 1F5, hybridoma 3F2, hybridoma 15F11, hybridoma 17C2, orhybridoma 16G9. Therefore, according to another embodiment of thepresent invention, there is provided a composition for predicting ordiagnosing transplant rejection in a mammal, comprising the monoclonalantibody according to the present invention as active ingredient. To theabovementioned composition, pharmaceutically acceptable carriers can beadded, if desired. Further, the abovementioned transplant rejection ispreferably one which occurs after organ transplantation, more preferablyone which occurs after withdrawal of the administration of animmunosuppressive agent. Further, according to another embodiment of thepresent invention, there is provided use of the monoclonal antibodyaccording to the present invention as an agent for predicting ordiagnosing transplant rejection in a mammal. Further, according toanother embodiment of the present invention, there is provided a methodfor predicting or diagnosing transplant rejection in a mammal,comprising the measuring the level of immunoreactivity between abiological sample derived from a mammal and the monoclonal antibodyaccording to the present invention. In the abovementioned method, therisk of transplant rejection is predicted or diagnosed to be high whenthe measured level of immunoreactivity is higher than a threshold valuewhich is previously set referring to the level of immunoreactivitybetween a biological sample of a mammal suffering from transplantrejection and the monoclonal antibody according to the presentinvention. This threshold value is appropriately determined by thoseskilled in the art according to the species and sex of the mammal and adonor, the kind of transplant organ, the measuring method, and the like.According to the method for the prediction and the diagnosis accordingto the present invention, physical and financial burdens on a patientcan be reduced by avoiding the administration of unnecessaryimmunosuppressive agents.

Further, an example of the abovementioned biological sample ispreferably blood, more preferably serum.

The abovementioned immunosuppressive agents are not particularly limitedas long as they are immunosuppressive agents used in organtransplantation. They can be alkylating agents such as cyclophosphamide;anti-metabolites such as azathiopurine, methotrexate, and mizoribine;T-cell activity inhibitors such as cyclosporin and tacrolimus; steroidalagents such as prednisolone, methylprednisolone, mycophenolate mofetil,and azathiopurine; and lymphocyte surface function inhibitors such asbasiliximab and muromonab, or combinations thereof.

The abovementioned mammal and transplant organ donor can be, forexample, humans, pigs, and baboons, preferably humans. Examples of theorgan to be transplanted are the liver, heart, kidney, and skin.

A method of measuring the abovementioned level of immunoreactivity isnot particularly limited as long as it utilizes antigen-antibodyreaction; specific examples of the method include the fluorescenceantibody method, the chemical staining method, the enzyme-antibodymethod, the ELISA method, radioimmunoassay, immunoprecipitation, theWestern blot method, modified Western blot methods (e.g., the Westernmethod, the Southwestern method, the Northwestern method, andWest-western method), and the protein chip method. Therefore, accordingto another preferred embodiment of the present invention, theabovementioned level of immunoreactivity is measured by the fluorescenceantibody method, the chemical staining method, the enzyme-antibodymethod, the ELISA method, radioimmunoassay, the immunoprecipitationmethod, the Western blot method, a modified Western blot method, or theprotein chip method.

Further, according to another embodiment of the present invention, thereis provided a kit for predicting or diagnosing transplant rejection in amammal, comprising at least an anti-histone H1 monoclonal antibody. Theabovementioned transplant rejection is preferably one which occurs afterorgan transplantation, more preferably one which occurs after withdrawalof the administration of an immunosuppressive agent.

Polypeptides

A polypeptide according to the present invention comprises theabovementioned epitope which is recognized by the anti-histone H1monoclonal antibody according to the present invention, in its aminoacid sequence. Further, according to a preferred embodiment of thepresent invention, the polypeptide consists of an amino acid sequencerepresented by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,or SEQ ID NO: 8.

Also, according to another preferred embodiment of the presentinvention, the polypeptide consists of a modified amino acid sequencewhich includes substitutions, deletions, or addition of one or severalamino acids in an amino acid sequence represented by SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. As used herein,“one or several” generally ranges preferably about from 1 to 3, morepreferably about from 1 to 2.

Further, according to another preferred embodiment of the presentinvention, the polypeptide consists of a partial sequence of an aminoacid sequence represented by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, or SEQ ID NO: 8. Examples of the abovementioned partialsequence preferably include the partial sequence represented by aminoacid number 6 to 9 in SEQ ID NO: 4, the partial sequence represented byamino acid number 5 to 9 in SEQ ID NO: 5, the partial sequencerepresented by amino acid number 2 to 5 in SEQ ID NO: 6, the partialsequence represented by amino acid number 2 to 5 in SEQ ID NO: 7, andthe partial sequence represented by amino acid number 7 to 9 or aminoacid number 11 to 12 in SEQ ID NO: 8.

Further, according to another embodiment of the present invention, thepolypeptide comprises an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or apartial sequence thereof. Further, this polypeptide consists ofpreferably about 3 to 300, more preferably about 12 to 150 amino acidresidues.

The amino acid sequence of the polypeptide according to the presentinvention is determined based on the analysis using a phage displaypeptide library kit with the monoclonal antibody according to thepresent invention. The polypeptide according to the present inventioncan be synthesized based on this amino acid sequence using a knownpeptide synthesizing device and the like.

Further, the polypeptide according to the present invention can be usedfor inducing the production of an anti-histone H1 antibody in the body,as is or after derivertizing it by a known method. Further, histone H1or a histone H1-like antigen can also exhibit an immunosuppressiveactivity by administering it to the body as a sensitizing antigen toproduce an anti-histone H1 antibody. Therefore, according to a preferredembodiment of the present invention, there is provided a composition forsuppressing immunity comprising histone H1, a histone H1-like antigen,or a polypeptide according to the present invention as activeingredient. Further, according to another embodiment of the presentinvention, there is provided use of histone H1, a histone H1-likeantigen, or a polypeptide according to the present invention inmanufacturing the composition for suppressing immunity.

The abovementioned composition for suppressing immunity can bemanufactured in a dosage form suited to its administration method alongwith pharmaceutically acceptable carriers. For example, when its dosageform is a fluid, appropriate solvents, solubilizing agents,preservatives, stabilizers, emulsifiers, suspending agents, analgesicagents, isotonizing agents, buffering agents, fillers, thickeningagents, coloring agents, known carriers (e.g., various liposomes,polyamino acids, carriers, synthetic polymers, natural polymers),adjuvants, and the like can be appropriately contained.

A method for administrating the abovementioned composition forsuppressing immunity can be any method usable in this field oftechnology, including intra-arterial injection, dripping, intravenousinjection, intramuscular injection, subcutaneous injection, intradermalinjection, oral administration, mucosal administration, and transdermaladministration. Therefore, according to another embodiment of thepresent invention, there is provided a method for treating a mammalwhich requires immunosuppression, comprising administering atherapeutically effective amount of histone H1, a histone H1-likeantigen, or a polypeptide according to the present invention, to amammal. The therapeutically effective amount varies depending on theseverity of the symptoms, sex, age, bodyweight, behavior of the mammal;however, the amount of the active ingredient can be 0.005 μg to 2 g/kgbodyweight/day. Further, its medication regimen can be appropriatelymade by those skilled in the art by confirming antibody production inthe mammal.

Further, histone H1, a histone H1-like antigen, or a polypeptideaccording to the present invention can be used in measuring the amountof anti-histone H1 antibody in a mammal since it specifically reactswith an anti-histone H1 antibody produced in the mammal. Therefore,according to another embodiment of the present invention, there isprovided a composition for measuring the amount of anti-histone H1antibody in a biological sample derived from a mammal, comprisinghistone H1, a histone H1-like antigen, or a polypeptide according to thepresent invention as active ingredient. Since the abovementionedanti-histone H1 antibody has a function to suppress transplant rejectionas mentioned above, the amount of anti-histone H1 antibody in the mammalmakes an index for the prediction and diagnosis of transplant rejection.Therefore, according to another preferred embodiment of the presentinvention, the abovementioned composition can be used for the predictionand diagnosis of transplant rejection in a mammal. Further, according toanother embodiment of the present invention, there is provided use ofhistone H1 according to the present invention, a histone H1-likeantigen, or a polypeptide according to the present invention as agentfor predicting or diagnosing transplant rejection in a mammal. Theabovementioned transplant rejection is one which occurs after organtransplantation, more preferably one which occurs after withdrawal ofthe administration of an immunosuppressive agent. Further, according toanother embodiment of the present invention, there is provided a methodfor predicting or diagnosing transplant rejection in a mammal,comprising measuring the level of immunoreactivity between ananti-histone H1 antibody in a biological sample in a mammal and histoneH1, a histone H1-like antigen, or a polypeptide according to the presentinvention. In the abovementioned method, the risk of transplantrejection is predicted or diagnosed to be low when the measured level ofimmunoreactivity is higher than a threshold value which is previouslyset referring to the level of immunoreactivity between the anti-histoneH1 antibody of a biological sample from a mammal suffering fromtransplant rejection and histone H1, a histone H1-like antigen, or apolypeptide according to the present invention. This threshold value isappropriately determined by those skilled in the art according to thespecies and sex of the mammal and a donor, the kind of transplant organ,the measuring method, and the like. According to the method of theprediction or diagnosis according to the present invention, physical andfinancial burdens on a patient can be reduced by avoiding theadministration of unnecessary immunosuppressive agents.

A method for measuring the abovementioned level of immunoreactivity isnot particularly limited as long as it uses the antigen-antibodyreaction; specific examples of the method include the fluorescenceantibody method, the chemical staining method, the enzyme-antibodymethod, the ELISA method, radioimmunoassay, immunoprecipitation, theWestern blot method, or modified Western blot methods (e.g., the Westernmethod, the Southwestern method, the Northwestern method, theWest-western method), and the protein chip method. The abovementionedimmunoreactivity level can be measured preferably by the fluorescenceantibody method, the chemical staining method, the enzyme-antibodymethod, the ELISA method, radioimmunoassay, the immunoprecipitationmethod, the Western blot method, a modified Western blot method, or theprotein chip method, more preferably the protein chip method. In theprotein chip method, the prediction or diagnosis of transplant rejectionin a mammal can be carried out rapidly and accurately by loading apolypeptide according to the present invention onto a protein chip.

Further, according to another embodiment of the present invention, thereis provided a kit for measuring the amount of anti-histone H1 antibodyin a biological sample derived from a mammal, comprising at leasthistone H1, a histone H1-like antigen, or a polypeptide according to thepresent invention. Further, according to another preferred embodiment ofthe present invention, the abovementioned kit is for predicting ordiagnosing transplant rejection in a mammal. The abovementionedtransplant rejection is preferably one which occurs after organtransplantation, more preferably one which occurs after withdrawal ofthe administration of an immunosuppressive agent.

The abovementioned biological samples, immunosuppressive agents,transplant organs, mammals, transplant organ donors, and the like arethe same as in the therapeutic method and the method of predicting ordiagnosing transplant rejection using the monoclonal antibody accordingto the present invention.

EXAMPLES

The following examples will specifically explain the present invention;however, they are not to be construed to limit the scope of theinvention.

Reagents and antibodies used were of analytical grade, unless otherwisementioned. Further, DA rats and PVG rats (males, 7 to 8 weeks of age)were purchased from Japan SLC, Inc and Seac Yoshitomi, Ltd.,respectively. BALB/c mice (males, 5 to 6 weeks of age) were purchasedfrom Charles River Japan, Inc.

Reference Example 1 Mixed Lymphocyte Reaction (MLR): Rat Cells

Splenic lymphocytes derived from untreated PVG rats (responding cells)and splenic lymphocytes derived from DA rats treated with mitomycine C(Kyowa Hakko Kogyo Co., Ltd.) (stimulating cells) were used. Theresponding cells were adjusted to 5×10⁵ cells/mL with a 10% FCS-RPMImedium, and the stimulating cells were adjusted to 8×10⁶ cells/mL with a10% FCS-RPMI medium. The responding cell suspension and the stimulatingcell suspension thus prepared were seeded each in a 100 μL portion ontoa 96-well round bottom plate (Nunc Brand Products), after which at thestart of mixed cultivation, anti-histone H1 polyclonal IgG (Santa CruzBiotechnology, at 0.1, 0.2, 0.4, 0.8, or 1.6 μg/well) or rabbit IgG(normal rabbit IgG, Santa Cruz Biotechnology, at 0.1, 0.2, 0.4, 0.8, or1.6 μg/well) were added and cultivation was carried out at 37° C. in anatmosphere of 5% CO₂/95% air for more than 3.5 days. Here, tacrolimus(FK506, Fujisawa Pharmaceutical Co., Ltd.) was added as a positivecontrol. Further, 15 hours prior to completion of the cultivation, 10 μLof bromodeoxyuridine (BrdU) was added. Then, using a BrdU labeling &detection kit III (Roche Diagnostics), the degree of cell proliferationwas determined using the amount of BrdU incorporated into intracellularDNA as an index. Here, the higher the degree of cell proliferation, themore BrdU is incorporated.

As a result, when anti-histone H1 polyclonal IgG was added, MLR wassignificantly inhibited and the inhibition level was the same as that ofthe case that tacrolimus was added.

Reference Example 2 Mixed Lymphocyte Reaction (MLR): Human Cells

Preparation of Lymphocytes

From two humans (A and B), 10 mL each of peripheral blood was taken andcentrifuged (1500 rpm, 30 minutes), after which the plasma was removed.Next, PBS in the same volume as the removed plasma (3 mL) was added tothe residue and the admixture was stirred. To this admixture, 3 ml ofFicoll-paque solution (Amersham Biosciences) was added and the resultingadmixture was subjected to density gradient centrifugation (1500 rpm, 30minutes) to obtain a white middle layer containing lymphocytes. Thetotal volume of 12 mL of cell suspension was made by adding sterilizedPBS to this white layer and subjected to centrifugation (1500 rpm, 5minutes). After repeating this procedure twice, the lymphocytes obtainedwere suspended in 1 mL of 10% FCS-AIM-V medium (GIBCO).

MLR

The following test was carried out using lymphocytes derived from B(responding cells) and lymphocytes derived from A treated with mitomycinC (Kyowa Hakko Kogyo, Co., Ltd.) (stimulating cells). The respondingcells were adjusted to 5×10⁵ cells/mL with a 10% FCS-AIM-V medium, andthe stimulating cells were adjusted to 8×10⁶ cells/mL with a 10%FCS-RPMI medium. The responding cell suspension and the stimulating cellsuspension thus prepared were seeded each in a 100 μl portion onto a96-well round bottom plate (Nunc Brand Products), after which at thestart of mixed cultivation, anti-histone H1 polyclonal IgG (Santa CruzBiotechnology, at 0.1, 0.2, 0.4, 0.8, or 1.6 μg/well) or rabbit IgG(normal rabbit IgG, Santa Cruz Biotechnology, at 0.1, 0.2, 0.4, 0.8, or1.6 μg/well) was added and cultivation was carried out at 37° C. in anatmosphere of 5% CO₂/95% air for 2.5 days. Here, tacrolimus was added asa positive control. Further, during cultivation, ConA was added to eachwell at a final concentration of 10 μl/mL to confirm that theproliferation of the stimulating cells was stopped and that theresponding cells were proliferated by antigen stimulation. Then, eachwell was treated in the same manner as in Reference Example 1, and usinga BrdU labeling & detection kit III (Roche Diagnostics), the degree ofcell proliferation was determined using the amount of bromodeoxyuridine(BrdU) incorporated into intracellular DNA as an index.

In the combination of the responding cells derived from B and thestimulating cells derived from A treated with mitomycin C, cellproliferation by MLR was confirmed. Further, MLR was suppressedconcentration-dependently by the anti-histone polyclonal antibody. Onthe other hand, MLR was not suppressed by rabbit IgG (normal rabbitIgG).

Example 1 Preparation of Hybridomas

Immunization

A suspension (antigen concentration: 0.25 mg/ml) was obtained by mixing0.8 mL of a solution of an antigen (Histone H1 Histone F1 Histone KAP,Roche) in PBS (antigen concentration: 0.5 mg/mL) and 0.8 mL of Freund'scomplete adjuvant (Wako Pure Chemical Industries). Next, 0.2 mL of thissuspension was intraperitoneally administered to a BALB/c mouse.Further, an equal amount of this suspension was administered to themouse every 2 weeks. Then, 16 weeks after the start of administration,0.2 mL of an antigen solution in PBS (antigen concentration: 600 to 1000mg/mL) was lastly administered intraperitoneally to the mouse. Uponadministration, blood was collected from the ocular fundus vein and theantibody titer was measured by ELISA. Four days after the finaladministration, whole blood was collected and the blood obtained wascentrifuged (2000 rpm, 20 minutes) to obtain antiserum which was used asa control antiserum in the following experiment. Further, after wholeblood was collected, the spleen was dissected from a rat and spleencells obtained were used in the following cell fusion.

Cell Fusion

The abovementioned spleen cells and myeloma cells (P3×63-Ag.8.653) weremixed at a ratio of 10:1 to 10 and the mixture was centrifuged (1500rpm, 5 minutes). After centrifugation, the supernatant was removed usingan aspirator and to the resulting cell pellet, 1 mL of polyethyleneglycol 4000 (50% solution in PBS) at 37° C. was added over a period ofone minute to make a mixed solution. This mixed solution was allowed tostand at 37° C. for one minute, after which an IMDM medium (total 9 ml)at 37° C. was added at a rate of 1 mL per 30 seconds and then theadmixture was centrifuged (1500 rpm, 5 minutes). After centrifugation,the supernatant was removed by suction and an appropriate amount of anIMDM medium (GIBCO) supplemented with 15% FCS (JRH BIOSCIENCES) at 37°C. was added. A 100 mL aliquot of the resulting suspension was eachdispensed onto a 96 well culture plate and the plate was incubated at37° C. for one day in a 5% CO₂ incubator. Further, 100 mL of an HATmedium (which was prepared by dissolving HAT powder (HAT MEDIASUPPLEMENT (×50, Sigma) in 10 mL of a serum-free IMDM medium anddiluting the solution 50 times with an IMDM medium containing 10% FCS)was added and incubation was carried out at 37° C. in a 5% CO₂incubator. The HAT medium was changed every 2 to 3 days. After 10 days,the medium was changed to an HT medium (which was prepared by dissolvingHT powder (HT MEDIA SUPPLEMENT, Sigma) in 10 mL of a serum-free IMDMmedium and diluting the solution 50 times with an IMDM medium containing10% FCS) and incubation was carried out at 37° C. for 3 days in a 5% CO₂incubator. Then, the medium (HT medium) was changed every 2 to 3 days.After confirming cell proliferation under a microscope, the culturesupernatant (about 100 mL) was recovered. Using this culturesupernatant, hybridoma screening was carried out by measuring theantibody titer against histone H1 as shown below.

Screening of Hybridoma Cells

Measurement of Antibody Titer

An aliquot of 50 μL per well of a buffer solution containing histone H1(5 mg, calf thymus histone H1, Roche Diagnostics) (bicarbonate buffer:100 mM NaHCO₃—NaOH, pH 9.2-9.5; histone H1 concentration: 1 mg/mL) wasdispensed onto a 96-well flat bottom plate and the plate was allowed tostand at room temperature for 2 hours for coating. The plate was washed3 times with a washing buffer solution (PBST), a blocking buffersolution (3% skim milk and 1% BSA in PBS) was added at 200 to 250μL/well, and after reacting at 4° C. for 24 hours, the plate was washed3 times. Next, a 100 μL aliquot per well of the hybridoma culturesupernatant was added and the reaction was carried out at 37° C. for 4hours or at 4° C. for 24 hours. After washing the plate 3 times, a 50 μLaliquot per well of biotin-labeled anti-mouse IgG (Sigma) which wasdiluted 10000 times with a dilution buffer solution (10 mM Tris-HCl (pH8.0), 0.9% (W/V) NaCl, 0.05% (W/V) Tween 20) was added and the reactionwas carried out at room temperature for 2 hours. After washing 6 times,a 50 μL aliquot per well of alkaline phosphatase-labeled streptavidinwhich was diluted 1000 times with a dilution buffer was added and thereaction was carried out at room temperature for 1 to 2 hours. Afterwashing 6 times, a 50 μL aliquot per well of a fluorescent substratebuffer (Attophos substrate buffer, Roche Diagnostics) was added todevelop color while protecting the plate from light. Fluorescence wasmeasured by CytoFlour II (PerSeptive).

Selection of Hybridomas

An IMDM medium supplemented with 15% FCS and 10% HCF (hybridoma cloningfactor, Origen) was added to the wells showing positive reaction in theabovementioned antibody titer measurement (1×10⁵ cells/mL), theresultant suspension was dispensed onto a 96-well culture plate to makethe cell concentration about 200 cells/well, and the incubation wascarried out at 37° C. in a 5% CO₂ incubator. Then, the antibody titermeasurement was carried out in the same manner as mentioned above toselect hybridomas having high antibody productivity.

Further, limiting dilution was carried out with an IMDM mediumsupplemented with 15% FCS and 10% HCF to make the selected hybridomaconcentration 0.5 to 1 cell/well and the incubation was carried out at37° C. in a 5% CO₂ incubator for about 3 to 4 days, after which theantibody titer was measured in the same manner as described above toselect hybridomas having high antibody productivity. Further, thelimiting dilution was further repeated to obtain 38 hybridomas producinganti-H1 monoclonal antibody. From these hybridomas, 10 hybridomasshowing higher antibody titer than the control antiserum were selectedand designated 1F5, 3F2, 15A11, 15F11, 16D1, 16G9, 17C2, 17E2, 21E3, and22A12.

Test Example 1 Test for Evaluating MLR Suppressing Activity by CultureSupernatant Containing Anti-Histone H1 Monoclonal Antibody

Preparation of Culture Supernatant Containing Anti-Histone H1 MonoclonalAntibody

Each hybridoma was cultured using an IMDM medium supplemented with 15%FCS and 10% HSF (1×10⁶ cells/mL). Using CENTRIPREP YM-10 (MILLIPORE), 15mL of this culture supernatant was centrifuged (2000 g, 2.5 hours) toobtain a culture supernatant concentrate containing anti-histone H1monoclonal antibody.

MLR

Using the culture supernatant concentrate obtained (a 1000-fold dilutedsolution), the MLR suppressing activity evaluation test was carried outin the same manner as in Reference Example 1. As controls, rat serumbefore immunization (1000-fold diluted solution), rabbit IgG (normalrabbit IgG, 1.6 mg, Santa Cruz Biotechnology) at 1.6 μg/well,anti-histone H1 polyclonal IgG (200 μg, Santa Cruz Biotechnology) at 1.6μg/well, and tacrolimus at 1.6 μg/well were used. An IMDM medium wasused for dilution.

Results are shown in FIG. 1. The culture supernatants of 8 hybridomas,1F5, 3F2, 15A11, 15F11, 16D1, 16G9, 17C2, and 17E2, showed an MLRsuppressing activity as equivalent to anti-histone H1 polyclonalantibody and tacrolimus. In FIG. 1, PVG/PVG represents a mixed cultureof PVG lymphocytes which lost proliferating ability by mitomycin Cstimulation with PVG lymphocytes which were responding cells, and DA/PVGrepresents a mixed culture of DA lymphocytes which similarly lostproliferating ability with PVG lymphocytes which were responding cells.

Test Example 2 Specification of Recognition Site of Anti-Histone H1Monoclonal Antibody 1

Preparation of Samples for Measurement

Spleen cells were dissected from PVG rats and subjected to celldisruption according to the method of Weissman (Weissman et al., Science239, 1018-1021, 1988). The spleen cells were centrifuged with 1 mL ofPBS (1500 rpm, 5 minutes) and then recovered. These cells were made intosuspension using a syringe and an equal volume of 150 mM NaCl solutionwas added. The resulting cell suspension was centrifuged (300×g, 10minutes) to obtain the precipitate and supernatant. This precipitate wasdesignated as an insoluble fraction (containing a nuclear fraction) andthe supernatant was designated as a soluble fraction containing cellmembranes. To the insoluble fraction, a 5-fold volume (v/v) of samplebuffer for electrophoresis (25 mL of 0.25 M Tris-HCl (pH 6.8), 2.0 g ofSDS, 9 mL of ultra-pure water, 10 mL of glycerol, 5 mg of BPB) wasadded. The resulting admixture was boiled for 5 minutes and thencentrifuged to obtain a supernatant for use as a sample for measurement.Further, to the soluble fraction containing cell membranes, EDTA wasadded at a final concentration of 5 mM. Of this soluble fractioncontaining cell membranes, an aliquot of 100-200 μL was recovered. Theremaining soluble fraction containing cell membranes was subjected toultracentrifugation (4° C., 200000×g, 45 minutes). The resultingprecipitate was designated as a cell membrane fraction and the resultingsupernatant was designated as a cell membrane-free soluble fraction. Thecell membrane fraction was boiled for 5 minutes with a 5-fold volume(v/v) of sample buffer for electrophoresis, and then centrifugation wascarried out. The supernatant thus obtained was used as a sample formeasurement.

Further, 10 mL of the culture supernatant containing anti-histone H1monoclonal antibody obtained in Test Example 1 was diluted with abinding buffer (20 mM phosphate buffer, pH 7.4) to obtain 50 mL of asolution. This solution was loaded onto a HiTrap Protein G column(HiTrap Protein G HP, Amersham Biosciences) at a flow rate of 0.2-1mL/min and circulated at 4° C. for 24 hours. Then, 5 mL of bindingbuffer was passed through at a flow rate of 1 to 2 mL/min to wash thecolumn. After washing the column, the abovementioned soluble fractionsolution containing cell membranes was loaded at a flow rate of 0.2 to 1mL/min and circulated at 4° C. for 24 hours. Next, 2 mL of bindingbuffer was passed through at a flow rate of 1 to 2 mL/min to obtain aflow-through fraction. This flow-through fraction was used as a samplefor measurement.

Purification of Anti-Histone H1 Monoclonal Antibody

1 mM HCl solution was loaded onto a HiTrap NHS column (HiTrapNHS-activated HP, Amersham Biosciences AB) at a flow rate of 1 tp 2mL/min and then 1 mL of a histone H1 solution (10.5 mg of histone H1(Roche Diagnostics), coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3))was loaded at a flow rate of 1 mL/min, after which the column wasimmediately sealed to carry out coupling (15 to 30 minutes). Aftercoupling, the inside of the column was washed with buffer A (0.5 Mmonoethanolamine, 0.5 M NaCl, pH 8.3), buffer B (0.1 M sodium acetate,0.5 M NaCl, pH 4.0), and neutral buffer (1.0 M Tris-HCl, pH 9.0). Next,the culture supernatant containing anti-histone H1 monoclonal antibodyobtained in Test Example 1 was loaded onto the column at a flow rate of1 mL/min and circulated (4° C., overnight). Next, after washing thecolumn with 5 mL of phosphate buffer, 5 mL of elusion buffer was passedthrough at a flow rate of 0.2 to 1 mL/min and a purified anti-histone H1monoclonal antibody was obtained from the eluted fraction.

SDS-PAGE

According to electrophoresis in the discontinuous buffer system byLaemmli (Nature, 227, 680-685, 1970), each sample for measurement wastreated by SDS-PAGE. As a control, histone H1 (5 mg, Roche Diagnostics)was used. After SDS-PAGE, the resulting gel was subjected to thefollowing Coomassie staining or Western blotting.

Coomassie Staining

The gel after SDS-PAGE was immersed in a staining solution (0.25%Coomassie brilliant blue R/ethanol:acetic acid:distilled water=9:2:9)and shaken for about one hour, after which the gel was immersed in adestaining solution (ethanol:acetic acid:distilled water=25:8:65) forabout one hour for destaining. Then, the gel was immersed in apreservation solution (methanol:acetic acid:distilled water=10:15:175)to destain background.

Western Blotting

Proteins on the gel after SDS-PAGE were transferred onto a PVDF membraneusing a semidry-type transferring apparatus (AE-6675, ATTO). Next, thePVDF membrane after the transfer was immersed in a blocking solution (5%skim milk and 1% BSA in a PBST solution) and shaken (at 4° C. for 24hours or at room temperature for one hour). Next, a solution containingpurified anti-histone H1 monoclonal antibody (diluted 500 times with ablocking solution) was added to the PVDF membrane as a primary antibodyand after shaking at room temperature for one hour, the membrane waswashed with PBST once for 15 minutes and 3 times for 5 minutes. Afterwashing, a secondary antibody (HRP-anti-mouse IgG, Sigma) solutiondiluted 20000 times with a blocking solution was added to the PVDFmembrane and the membrane was shaken at room temperature for one hour.After shaking, the membrane was washed with PBST once for 15 minutes and3 times for 5 minutes. Further, binding of specific antibodies wasdetected using an ECL Plus Western blotting detection system (AmershamBiosciences AB) and developed after exposure to light using the X-rayfilm RX-U (FUJI PHOTO FILM, Tokyo, Japan).

As a result of Western blotting, a specific band was detected at aposition of 31 kD of the cell membrane fraction. This band was detectedat the same position as histone H1 in Coomassie staining.

Test Example 3 Specification of Recognition Site of Anti-Histone H1Monoclonal Antibody 2

The PVG rat spleen cells were suspended in a PBS solution containing 4%formalin and fixed at room temperature for 20 minutes. Further, thespleen cells were washed 3 times with a staining buffer (a PBS solutioncontaining 1% (v/v) FCS and 0.1% (w/v) sodium azide, 4° C.), after whicha mixed solution containing 2×10⁶ cells in 100 ml of staining buffer wasprepared. To this mixed solution, a primary antibody (2 mL ofbiotin-labeled anti-histone H1 monoclonal antibody or 5 mL ofbiotin-labeled normal mouse IgG) was added and the reaction was carriedout at 37° C. for one hour. After this reaction, the spleen cells werewashed with a staining buffer (4° C.) 3 times, further 100 mL ofstaining buffer and 1 mL of FITC-labeled streptavidin (BD PharMingen)were added to the spleen cells and the reaction was carried out at roomtemperature for 30 hours. After the reaction, the spleen cells werewashed 3 times with staining buffer (4° C.), and 500 mL of PBS was addedto the spleen cells to prepare a suspension. Further, propidium iodide(Sigma) was added to this suspension at a final concentration of 5 mg/mLand the reaction was carried out at room temperature for 20 minutes. Thecells thus obtained were sealed with a PBS solution containing 50%glycerin and then examined under a fluorescent microscope.

As a result, only the peripheral part (cell membrane) of the spleencells was specifically fluorescence-stained.

Test Example 4 Specification of Recognition Site of Anti-Histone H1Monoclonal Antibody 3

Phage Display

For the anti-histone H1 antibody produced by hybridoma 1F5, 3F2, 15F11,17C2 or 16G9, a panning experiment was carried out using the Ph.D.-12phage display peptide library kit (purchased from New England BioLabs,Inc.). Each purified monoclonal antibody dissolved in a 0.1 M NaHCO₃solution (pH 8.6) was used for direct coating onto a microtiter plate(Nunc, catalog #430341) and the plate was incubated at 4° C. overnight.Blocking buffer (0.1 M NaHCO₃, 5 mg/mL BSA, 0.02% NaN₃) was added toeach well and the plate was incubated at 4° C. for at least one hour andthen washed with TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween 20). In thefirst panning, 4×10¹⁰ phages in the original library were used forscreening. Unbound phages were removed by repetitive washing with TBTS.Bound phages were eluted with 0.2 M glycine-HCl buffer (pH 2.2, 1 mg/mLBSA). The phages eluted were proliferated in 20 mL of E. coli ER2738culture. The resulting phages were precipitated using polyethyleneglycol and used for the second panning. Further, the third panning wascarried out according to the same procedure. Plaques obtained in thethird panning were diluted 100 times and were proliferated using ER2738culture. Tubes containing the resulting product were incubated withshaking at 37° C. for 4.5 to 5 hours. A single-stranded phage DNA wasprecipitated and purified using iodide buffer (10 mM Tris-HCl, 1 mMEDTA, 4 M NaI) and ethanol. The phage DNA was dissolved in 20 μl of TEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) for DNA sequence analysis.

DNA Sequence Analysis

For the purified phage DNA obtained, sequencing PCR reaction was carriedout using primer DNA attached to the abovementioned Ph.D.-12 phagedisplay peptide library kit and the DYEnamic™ ET Terminator CycleSequencing Premix Kit (Amersham Biosciences) (PCR reaction conditions:30 cycles consisting of 30 seconds at 95° C., 15 seconds at 50° C., andone minute at 60° C.). The PCR products were purified using the AutoSeg™G-50 (Amersham Biosciences). Further, the DNA sequence of each of thephage peptide was determined using the ABI PRISM™ 310 Genetic Analyzer(PE Biosystems). Amino acid sequences based on the determined DNAsequences are as follows:

Hybridoma strain  Amino acid sequence 1F5 NYQTYTPRPPHS (SEQ ID NO: 4)3F2 VTNNQTSPRWEI (SEQ ID NO: 5) 15F11 WKPVSLTLHTHP (SEQ ID NO: 6) 17C2HATGTHGLSLSH (SEQ ID NO: 7) 16G9 SSVLYGGPPSAA (SEQ ID NO: 8)

Competition ELISA

Each peptide having the amino acid sequence determined from theabovementioned phage DNA was synthesized according to an ordinarymethod. Competition ELISA was performed using the peptides obtained,each purified monoclonal antibody, and histone H1 antigen (Roche,catalog #1004875). Here, an EZ-Link Sulfo-NHS-Biotinylation kit (Pierce)was used for biotinylation of histone H1 antigen and an ABTS solution(Sigma, A3219) was used as a color forming reagent. Color formation wasdetected at 405 nm using an ELISA measuring apparatus (ThermoLabsystems, Multiskan Ascent). Measurements were made 3 times to obtainthe average of absorption values.

As a result, the abovementioned synthetic peptides were confirmed toinhibit the binding between each purified monoclonal antibody andhistone H1 antigen.

1. A monoclonal antibody which recognizes histone H1 or a histoneH1-like antigen present in the cell membrane of a spleen cell, whereinthe monoclonal antibody specifically recognizes an amino acid sequencerepresented by SEQ ID NO:
 8. 2. The monoclonal antibody according toclaim 1, which is produced by hybridoma 16G9.
 3. A pharmaceuticalcomposition, comprising the monoclonal antibody according to claim 1 anda pharmaceutically acceptable carrier.
 4. A diagnostic kit, comprisingat least the monoclonal antibody according to claim 1 and a detectionreagent thereof.
 5. A hybridoma which produces a monoclonal antibodywhich recognizes histone H1 or a histone H1-like antigen present in thecell membrane of a spleen cell, wherein the monoclonal antibodyspecifically recognizes an amino acid sequence represented by SEQ ID NO:8.
 6. The hybridoma according to claim 5, wherein the hydridoma ishybridoma 16G9.